Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

Harunori Yoshikawa, Mark Larance, Dylan J. Harney, Ramasubramanian Sundaramoorthy, Tony Ly, Thomas Owen-Hughes, Angus I. Lamond (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    32 Citations (Scopus)
    201 Downloads (Pure)

    Abstract

    We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

    Original languageEnglish
    Article numbere36530
    Pages (from-to)1-26
    Number of pages26
    JournaleLife
    Volume7
    Issue number7
    DOIs
    Publication statusPublished - 10 Aug 2018

    Keywords

    • Amino Acids/chemistry
    • Animals
    • Cell Line
    • Chromatography, High Pressure Liquid
    • Humans
    • Mice
    • Polyribosomes/chemistry
    • Protein Biosynthesis
    • Proteomics
    • Ribosomes/chemistry

    ASJC Scopus subject areas

    • General Biochemistry,Genetics and Molecular Biology
    • General Immunology and Microbiology
    • General Neuroscience

    Fingerprint

    Dive into the research topics of 'Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC'. Together they form a unique fingerprint.

    Cite this